bluuebunny/crossref_metadata_2025 huggingface dataset:
Developments of an efficient global optimal design technique – a combined approach of MLS and SA algorithm
Authors: Ho S.L. , Yang Shiyou , Ni Peihong , Wong H.C.
Published: October 2002

A new response surface model (RSM), the moving least squares (MLS) approximation, is proposed for reconstructing the objective/constraint functions for the design optimization of electromagnetic devices. The reconstructed functions are then combined with the simulated annealing (SA) algorithm to develop a computationally efficient technique to obtain the global solutions. The new method has: the “intelligence” to arrange the sample points, i.e. intensify the sample points in regions where a local optimum is likely to exist; the flexibility in dealing with irregular sample points; the self‐adaptive ability to regulate the parameters of the MLS models. Detailed numerical examples are given to validate the proposed technique.
Achieving breakthrough performance improvement: results of implementing a fit‐for‐purpose total management system
Authors: Chan Y.K. , Gaffney P. , Neailey K. , Ip W.H.
Published: October 2002

The Mass Transit Railway Corporation Ltd (MTR) of Hong Kong implemented a total management system (TMS) in its operations engineering department in 1998. In view of the many benefits brought about by TMS upon its implementation, the system has extended to cover the railway operations since 1999. Comparing performance before and after the implementation of TMS, almost all balanced scorecard indicators have shown improvement well beyond the target levels. This paper highlights the performance differences before and after the implementation of the TMS.
Randomized controlled trial of the Alexander Technique for idiopathic Parkinson's disease
Authors: Stallibrass C , Sissons P , Chalmers C
Published: October 2002

Objective: To determine whether the Alexander Technique, alongside normal treatment, is of benefit to people disabled by idiopathic Parkinson's disease.

Design: A randomized controlled trial with three groups, one receiving lessons in the Alexander Technique, another receiving massage and one with no additional intervention. Measures were taken pre and post-intervention, and at follow-up, six months later.

Setting: The Polyclinic at the University of Westminster, Central London.

Subjects: Ninety-three people with clinically confirmed idiopathic Parkinson's disease.

Interventions: The Alexander Technique group received 24 lessons in the Alexander Technique and the massage group received 24 sessions of massage.

Main outcome measures: The main outcome measures were the Self-assessment Parkinson's Disease Disability Scale (SPDDS) at best and at worst times of day. Secondary measures included the Beck Depression Inventory and an Attitudes to Self Scale.

Results: The Alexander Technique group improved compared with the no additional intervention group, pre-intervention to post-intervention, both on the SPDDS at best, p = 0.04 (confidence interval (CI) –6.4 to 0.0) and on the SPDDS at worst, p = 0.01 (CI –11.5 to –1.8). The comparative improvement was maintained at six-month follow-up: on the SPDDS at best, p = 0.04 (CI –7.7 to 0.0) and on the SPDDS at worst, p = 0.01 (CI –11.8 to –0.9). The Alexander Technique group was comparatively less depressed post-intervention, p = 0.03 (CI –3.8 to 0.0) on the Beck Depression Inventory, and at six-month follow-up had improved on the Attitudes to Self Scale, p = 0.04 (CI –13.9 to 0.0).

Conclusions: There is evidence that lessons in the Alexander Technique are likely to lead to sustained benefit for people with Parkinson's disease.
A self-administered pain severity scale for patellofemoral pain syndrome
Authors: Laprade J A , Culham E G
Published: October 2002

Objective: To develop a scale for estimating the severity of patellofemoral pain syndrome (PFPS) and to determine its reliability and validity.

Design: The PFPS Severity Scale (PSS) was developed following a literature search, input from clinicians, and pilot testing in people with PFPS. The final version of the instrument encompasses 10 statements regarding PFPS pain in a visual analogue format. Reliability and validity of the new scale were determined in a PFPS population.

Setting: All testing was performed at the Canadian Forces Base Kingston, Physiotherapy Department.

Subjects: Twenty-nine military subjects (7 female) between the ages of 20 and 48 (32 years ± 8.9) with subjective and objective findings consistent with PFPS were recruited. Twenty-four of the participants (6 female, 31.8 years ± 9.4) participated in the reliability phase of the study.

Methods: Reliability of the PSS was determined by comparing the scores obtained on two test days (24 hours apart). Convergent validity of the PSS was determined by comparing data from the PSS with two established knee scales: the WOMAC (Western Ontario and McMaster Universities) Osteoarthritis Index and the Hughston Foundation subjective knee scale. Results: Test–retest reliability was excellent (Spearman's rho = 0.95, p < 0.0001). The correlations between the PSS and the WOMAC and Hughston scales were strong (rho = 0.72 and 0.83, p < 0.001 respectively).

Conclusions: The PSS is reliable and has demonstrated convergent validity making it a useful tool for monitoring rehabilitative or surgical outcomes in clients with PFPS.
The effect of a first vaginal delivery on the integrity of the pelvic ‘ oor musculature
Authors: Marshall K , Walsh D M , Baxter G D
Published: October 2002

Objective: To assess the specific effect of delivery of the first child on the integrity of the pelvic floor musculature.

Design: A prospective study of two groups of females with no symptoms of urinary incontinence.

Setting: Physiotherapy Department, Rotunda Lying In Hospital, Dublin.

Subjects: Two groups of healthy female physiotherapists (age range 20–28 years) were recruited for the study: group 1 consisted of nulliparous females ( n = 10) and group 2 consisted of primiparous females who were 9–10 months post delivery ( n = 10).

Intervention and main outcome measures: Assessment of the pelvic floor musculature was performed by digital assessment, electromyography and perineometry.

Results: For all data, the nulliparous group showed evidence of greater pelvic floor strength and endurance. Analysis of anterior and posterior electromyography data showed significantly stronger contractions in the nulliparous group ( p = 0.0001 and 0.044). During a maximum contraction of the pelvic floor muscles, the anterior resting EMG activity increased by 9 ± 6 µV (mean ± SD) in the primiparous group compared with an increment of 22.3 ± 4.74 µV in the nulliparous group. Posterior EMG resting activity increased by 19.7 ± 7.65 µV (mean ± SD) in the nulliparous group compared with 13.8 ± 8.19 µV in the primiparous group. There were significant differences between the two groups for the four types of digital muscle assessment ( p < 0.0013). In addition, there was a greater increase in perineometry readings in the nulliparous group (increment = 5.6 ± 2.5, mean ± SD) compared with the primiparous group (increment = 3.1 ± 0.9; mean ± SD).

Conclusions: This study suggests that irrespective of lack of symptoms of urinary incontinence, it would appear advisable that all women should undertake a prescribed programme of pelvic floor rehabilitation exercises after childbirth.
The influence of aggregate microenvironment on the dissolution of oxazepam in ternary surfactant interactive mixtures
Authors: Stewart Peter J , Liu Julie
Published: October 2002

The purpose of this research was to test the hypothesis that the dissolution rate of oxazepam in interactive mixtures was dependent on the influence of surfactant within the microenvironment of mixed oxazepam-surfactant aggegrates produced during dissolution. The studies utilised both powder and intrinsic dissolution methodology; spectrophotometric assays were developed and validated and dissolution data were modelled using multi-exponential equations and dissolution rate constants estimated using non-linear least squares algorithms. For a series of water-soluble ternary additives to the oxazepam interactive mixture, sodium lauryl sulfate and cetrimide were shown not only to decrease aggregation through enhanced dispersion, but also to increase the dissolution rate constant. Such an increase in dissolution rate constant was observed in the intrinsic dissolution studies when surfactant concentrations exceeded the critical micelle concentration and the oxazepam solubility increased. Laser diffraction particle sizing during the dissolution process confirmed the presence of dispersed particles and aggregates and demonstrated that the presence of surfactant improved the state of dispersion. The results of studies using different rotational speeds produced unexpected increases in aggregation and decreases in dissolution rate constants at about 150 rev min−1, consistent with the transient formation of loose aggregates containing dissolved surfactant.
Intranasal administration of plasmid DNA-coated nanoparticles results in enhanced immune responses
Authors: Cui Zhengrong , Mumper Russell J
Published: October 2002

Intranasal immunization offers potential for the elicitation of effective mucosal and systemic immune responses. In this study, a previously reported novel cationic nanoparticle engineered from a microemulsion precursor was further modified, optimized and applied intranasally to mice to explore its potential as a plasmid DNA (pDNA) vaccine delivery system. To this end, more uniform nanoparticles (around 100 nm) containing less cationic surfactant were developed. The pDNA-coated nanoparticles significantly enhanced the specific serum IgG and IgA titres to an expressed model antigen, β-galactosidase, by 18–28 and 25–30 fold, respectively, when compared with naked pDNA alone. An enhanced splenocyte proliferative response was also observed after immunization with the pDNA-coated nanoparticles. It was concluded that these plasmid DNA-coated nanoparticles may have potential for immunization via the nasal route.
Synthesis of a psilocin hapten and a protein-hapten conjugate
Authors: Albers Christian , Lehr Matthias , Beike Justus , Köhler Helga , Brinkmann Bernd
Published: October 2002

Derivatives of psilocin with ω-functionalized alkyl spacers in position 1 of the indole ring were synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The protein–hapten conjugate was characterized by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4–5 molecules of psilocin hapten per molecule of BSA.
Byakangelicol, isolated from Angelica dahurica, inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells
Authors: Lin C H , Chang C W , Wang C C , Chang M S , Yang L L
Published: October 2002

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1β (IL-1β)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10–50 μm) concentration-dependently attenuated IL-1β-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01–1 μm), and byakangelicol (10–50 μm) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 μm, did not affect the activity and expression of COX-1 enzyme. IL-1β-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 μm), while byakangelicol (50 μm) had no effect. Treatment of cells with byakangelicol (50 μm) or pyrrolidine dithiocarbamate (PDTC; 50 μm) partially inhibited IL-1β-induced degradation of 1κB-α in the cytosol, translocation of p65 NF-κB from the cytosol to the nucleus and the NF-κB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1β-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1β-induced COX-2 expression may be, at least in part, through suppression of NF-κB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.
Analysis of glucose and lactate in dialysate from hypothalamus of rats after exhausting swimming using microdialysis
Authors: Wang L. , Dong Y. , Yu X. , Shangguan D. H. , Zhao R. , Han H. W. , Liu G. Q.
Published: October 2002

A microbore flow injection analysis–immobilized enzyme reactor–electrochemical detection (FIA‐IMER‐ECD) system for glucose and lactate detection was built up. The assays were precise, sensitive and practicable for determination of glucose and lactate levels in hypothalamic dialysate. The method had been used to detect the dynamic changes of glucose and lactate levels during rat exhausting swimming and recovery. The data showed that after exhausting swimming, the concentration of glucose in hypothalamic dialysate that reflected the concentration in the hypothalamic extracellular fluid decreased. The level fell to its nadir at day 1 after the exercise and then went back to the basal level at day 3 after the swimming. However, lactate levels increased to a maximum at day 3 and went back to the basal level at day 5 after the swimming. Copyright © 2002 John Wiley & Sons, Ltd.
CD44v10 interaction with Rho‐kinase (ROK) activates inositol 1,4,5‐triphosphate (IP3) receptor‐mediated Ca2+ signaling during hyaluronan (HA)‐induced endothelial cell migration
Authors: Singleton Patrick A. , Bourguignon Lilly Y.W.
Published: October 2002

Aortic endothelial cells (GM7372A) express a major cell adhesion molecule, CD44v10, which binds the extracellular matrix component, hyaluronan (HA), at its external domain and interacts with various signaling molecules at its cytoplasmic domain. In this study, we have determined that CD44v10 and Rho‐Kinase (ROK) are physically associated as a complex in vivo. Using a recombinant fragment of ROK (in particular, the pleckstrin homology [PH] domain) and in vitro binding assays, we have detected a specific binding interaction between the PH domain of ROK and the cytoplasmic domain of CD44. Scatchard plot analysis indicates that there is a single high‐affinity CD44 binding site in the PH domain of ROK with an apparent dissociation constant (Kd) of 1.76 nM, which is comparable to CD44 binding (Kd ∼1.56 nM) to intact ROK. These findings suggest that the PH domain is the primary ROK binding region for CD44. Furthermore, HA binding to GM7372A cells promotes RhoA‐mediated ROK activity, which, in turn, increases phosphorylation of three different inositol 1, 4, 5‐trisphosphate receptors (IP3Rs) [in particular, subtype 1 (IP3R1), and to a lesser extent subtype 2 (IP3R2) and subtype 3 (IP3R3)] all known as IP3‐gated Ca2+ channels. The phosphorylated IP3R1 (but not IP3R2 or IP3R3) is enhanced in its binding to IP3 which subsequently stimulates IP3‐mediated Ca2+ flux. Transfection of the endothelial cells with ROK's PH cDNA significantly reduces ROK association with CD44v10, and effectively inhibits ROK‐mediated phosphorylation of IP3Rs and IP3R‐mediated Ca2+ flux in vitro. The PH domain of ROK also functions as a dominant‐negative mutant in vivo to block HA‐dependent, CD44v10‐specific intracellular Ca2+ mobilization and endothelial cell migration. Taken together, we believe that CD44v10 interaction with ROK plays a pivotal role in IP3R‐mediated Ca2+ signaling during HA‐mediated endothelial cell migration. Cell Motil. Cytoskeleton 53:293–316, 2002. © 2002 Wiley‐Liss, Inc.
Dual diversity combining and decision feedback equalizer in indoor millimetre‐wave channel
Authors: Chiu Chien‐Ching , Hung Shi‐Cheng , Chen Chien‐Hung
Published: October 2002

Wideband communication characteristics of wireless indoor millimetre‐wave channel for arched and rectangular buildings are investigated. The impulse responses of arched and rectangular buildings for any transmitter–receiver location are computed by shooting and bouncing ray/image (SBR/Image) techniques. By using the impulse responses of these multipath channels, the impact of shapes of building is presented and the bit error rate performance of binary phase shift keying (BPSK) system with phase and timing recovery circuits are also calculated. Moreover, dual space antenna diversity technique and decision feedback equalizer (DFE) with four forward and three feedback taps are used to combat the multipath fading. Numerical results show that the mean root mean square (rms) delay spread for the arched building is smaller than that for the rectangular building. In addition, it is also found that the transmission rate can be up to 20 Mbps for indoor millimetre‐wave channel of these two buildings by using dual space diversity and DFE. Copyright © 2002 John Wiley & Sons, Ltd.
Attitudes towards prenatal diagnosis and termination of pregnancy among health professionals in Lebanon
Authors: Zahed L. , Nabulsi M. , Tamim H.
Published: October 2002

Objectives

To assess the attitudes of health professionals in Lebanon towards prenatal diagnosis and termination of pregnancy, for a series of genetic, non‐genetic and non‐medical conditions.

Methods

A total of 158 questionnaires were sent to geneticists, family doctors, pediatricians and obstetricians/gynecologists, that included information on sociodemographic variables and sets of questions and case scenarios, to which participants were asked to reply anonymously.

Results

Responses from the 75 participants revealed that the type of specialty did not significantly influence their attitude. However, acceptance of termination of pregnancy was influenced by gender, age, marital status, religion and its importance in their daily life. In general, acceptance of termination of pregnancy in the case of mild or severe clinical conditions was comparable to that reported from European countries, but more favorable in the case of sex chromosome abnormalities. Acceptance of prenatal diagnosis for non‐clinical conditions was, however, lower than that reported in Western nations.

Conclusion

The study provides a good basis for further studies with a larger number of respondents representing various geographical regions of the country. Copyright © 2002 John Wiley & Sons, Ltd.
Medial Smooth Muscle Thickness in Small Pulmonary Arteries in Sudden Infant Death Syndrome Revisited
Authors: Krous Henry F. , Floyd Catherine W. , Nadeau Julie M. , Silva Patricia D. , Blackbourne Brian D. , Langston Claire
Published: October 2002

Increased relative medial thickness (RMT) of smooth muscle in small pulmonary arteries, peripheral extension of smooth muscle into the alveolar wall arteries, and right ventricular hypertrophy (RVH), in response to purported prolonged hypoxia, have been reported in sudden infant death syndrome (SIDS). Prone sleep position, an important risk factor for SIDS, predisposes infants to hypoxia from airway obstruction or rebreathing. Since publication of the earlier pulmonary artery studies, the SIDS definition has been expanded, and sudden infant death investigational protocols have been implemented. Our aims in this study were to (1) compare RMT in preacinar arteries (PA), intra-acinar arteries accompanying small airways (SIA), and alveolar wall arteries (AW) in SIDS infants and controls; (2) correlate RMT with postmortem variables; (3) determine if peripheral extension occurred more often in SIDS infants than in controls; and (4) determine if RVH occurred in SIDS. Movat-stained sections from standardized tissue blocks taken prospectively from the apex of the right upper lobe from 88 SIDS cases and 17 controls were evaluated using a computer-assisted digitizing system with images obtained from a microscope with an attached video camera. When adjusted for age, the RMT values for the SIA arteries were significantly greater in controls, while the PA and AW arteries were not statistically different between the SIDS cases and controls. Peripheral medial smooth muscle extension did not differ between the groups, and RVH was not seen in SIDS cases. Given the recent identification of brain stem abnormalities interfering with protective cardiorespiratory responses against acute life-threatening hypoxia perhaps precipitated by prone sleeping, our data suggest that SIDS is an acute event not preceded by recurrent or prolonged apnea and hypoxia.
Three‐dimensional structure topology of the calreticulin P‐domain based on NMR assignment
Authors: Ellgaard Lars , Riek Roland , Braun Daniel , Herrmann Torsten , Helenius Ari , Wüthrich Kurt
Published: October 2002

Calreticulin (CRT) is an abundant molecular chaperone of the endoplasmic reticulum. Its central, proline‐rich P‐domain, comprising residues 189–288, contains three copies of each of two repeat sequences (types 1 and 2), which are arranged in a characteristic ‘111222’ pattern. Here we show that the three‐dimensional structure of CRT(189–288) contains a single hairpin fold formed by the entire polypeptide chain. The loop at the bottom of the hairpin consists of residues 227–247, and is closed by an anti‐parallel β‐sheet of residues 224–226 and 248–250. Two additional β‐sheets contain residues 207–209 and 262–264, and 190–192 and 276–278. The 17‐residue spacing of the β‐strands in the N‐terminal part of the hairpin and the 14‐residue spacing in the C‐terminal part reflect the length of the type 1 and type 2 sequence repeats. As a consequence of this topology the peptide segments separating the β‐strands in the N‐terminal part of the hairpin are likely to form bulges to accommodate the extra residues. These results are based on nearly complete sequence‐specific NMR assignments for CRT(189–288), which were obtained using standard NMR techniques with the 13C/15N‐labeled protein, and collection of nuclear Overhauser enhancement upper distance constraints.
Novel type Arabidopsis thaliana H+‐PPase is localized to the Golgi apparatus
Authors: Mitsuda Nobutaka , Enami Kazuhiko , Nakata Mami , Takeyasu Kunio , Sato Masa H.
Published: October 2002

Vacuolar H+‐PPase, a membrane bound proton‐translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants. Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H+‐PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome. We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen. Furthermore, the fluorescence of GFP‐tagged AVP2/AVPL1 showed small dot‐like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope. The distribution of this dot‐like fluorescent pattern was apparently affected by a treatment with brefeldin A. Moreover, we demonstrated that most dot‐like fluorescent structures colocalized with a Golgi resident protein. These findings suggest that this novel type H+‐PPase resides on the Golgi apparatus rather than the vacuolar membrane.
Use of HDEL‐tagged Trichoderma reesei mannosyl oligosaccharide 1,2‐α‐D‐mannosidase for N‐glycan engineering in Pichia pastoris
Authors: Callewaert Nico , Laroy Wouter , Cadirgi Hasan , Geysens Steven , Saelens Xavier , Min Jou Willy , Contreras Roland
Published: October 2002

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein‐linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human‐compatible N‐glycans in this organism is the conversion of the yeast‐type high‐mannose glycans to mammalian‐type high‐mannose and/or complex glycans. In this perspective, we have co‐expressed an endoplasmic reticulum‐targeted Trichoderma reesei 1,2‐α‐D‐mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans‐sialidase. Analysis of the N‐glycans of the two purified proteins showed a >85% decrease in the number of α‐1,2‐linked mannose residues. Moreover, the human‐type high‐mannose oligosaccharide Man5GlcNAc2 was the major N‐glycan of the glyco‐engineered trans‐sialidase, indicating that N‐glycan engineering can be effectively accomplished in P. pastoris.
UV Raman evidence of a tyrosine in apo‐human serum transferrin with a low pKa that is elevated upon binding of sulphate
Authors: Clarkson John , Smith D.Alastair
Published: October 2002

The binding of sulphate to human serum apo‐transferrin has been examined by ultraviolet absorption and ultraviolet resonance Raman difference spectroscopies between pH 6.0 and 9.0. The ultraviolet absorption data reveals a negative feature at 245 nm that increases in magnitude with pH, with an apparent pK
a of 7.57, which the Raman difference data reveals to be due to tyrosine. The pK
a of this tyrosine is unusually low and is measured at 7.84 by the Raman difference method and is elevated to greater than 9.0 upon addition of sulphate. Previous studies on the N‐lobe imply that Tyr 188 is the tyrosine with a low pK
a and also that Arg 124 is the primary binding site for the sulphate. The functional relevance may be that with sulphate bound, both carbonate binding and the deprotonation of Tyr will be disfavoured, and as a result so is iron binding.
A sensitive filter retention assay for the detection of PrPSc and the screening of anti‐prion compounds
Authors: Winklhofer Konstanze F , Hartl F.Ulrich , Tatzelt Jörg
Published: October 2002

A hallmark of prion diseases is the accumulation of an abnormally folded prion protein, denoted PrPSc. Here we describe a new and highly sensitive method for the detection of PrPSc in brain and other tissue samples that utilizes both PrPSc diagnostic criteria in combination; protease resistance and aggregation. Upon filtration of tissue extracts derived from scrapie‐ or bovine spongiform encephalopathy‐infected animals, PrPSc is retained and detected on the membranes. Laborious steps such as SDS–PAGE and Western blotting are avoided with concomitant gain in sensitivity and reliability. The new procedure also proved useful in a screen for anti‐prion compounds in a scrapie‐infected cell culture model.
A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase
Authors: Furtmüller Paul Georg , Jantschko Walter , Regelsberger Günther , Jakopitsch Christa , Moguilevsky Nicole , Obinger Christian
Published: October 2002

Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi‐mixing stopped‐flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9×107 M−1 s−1, pH 7 and 15°C) and reduction of compound I to compound II (3.0×104 M−1 s−1, pH 7 and 15°C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6×104 M−1 s−1, 1.4×106 M−1 s−1, 1.4×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively), whereas the endogenous one‐electron donor ascorbate mediated transformation of compound I to compound II (2.3×105 M−1 s−1) and of compound II back to the resting enzyme (5.0×103 M−1 s−1). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.
Molecular identification of phenylalanine ammonia‐lyase as a substrate of a specific constitutively activeArabidopsisCDPK expressed in maize protoplasts
Authors: Cheng Shu-Hua , Sheen Jen , Gerrish Chris , Bolwell G.Paul
Published: October 2002

Phenylalanine ammonia‐lyase (PAL) is a key enzyme in pathogen defence, stress response and secondary metabolism and is subject to post‐translational phosphorylation. In order to address the significance of this phenomenon it is necessary to identify the protein kinase (PK) responsible and place it in its regulatory circuit. Using protoplast transient expression ofArabidopsiskinase genes coupled to immunocomplex kinase assay, it has been possible to screen for specific PAL kinase. We show here that AtCPK1 (calcium dependent PK), but not other closely related PKs could phosphorylate both a recombinant PAL protein and a peptide (SRVAKTRTLTTA) that is a site phosphorylated in vivo. Identification of the specific CDPK as a PAL kinase now opens up the possibility of exploring the calcium link in biotic stress signalling, salicylate and phytoalexin production as well as the significance of PAL phosphorylation. The protoplast transient expression system is a potentially powerful method to determine and screen for plant gene functions utilising genomic and proteomic data.
Solid‐state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes
Authors: Bechinger Burkhard
Published: October 2002

Helical peptides reconstituted into oriented phospholipid bilayers were studied by proton‐decoupled 15N solid‐state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N‐terminal region of Vpu of HIV‐1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH‐dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail.
Perfusion‐induced redox differences in cytochrome c oxidase: ATR/FT‐IR spectroscopy
Authors: Nyquist Rebecca M. , Heitbrink Dirk , Bolwien Carsten , Wells Todd A. , Gennis Robert B. , Heberle Joachim
Published: October 2002

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction‐induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow‐exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time‐resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion‐induced Fourier‐transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.
Modulated kinase activities in cells undergoing tumour necrosis factor‐induced apoptotic cell death
Authors: Helms Matt J , Mohamed Ahmed A.A , MacEwan David J
Published: October 2002

Tumour necrosis factor‐α (TNF) has a variety of cellular effects including apoptotic and necrotic cytotoxicity. TNF activates a range of kinases, but their role in cytotoxic mechanisms is unclear. HeLa cells expressing elevated type II 75 kDa TNF receptor (TNFR2) protein, analysed by flow cytometry and Western analysis, showed altered c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (p38MAPK; but not MAPK) protein content and activation. There was greater JNK activation, but reduced p38MAPK activation in dying cells compared to those still to enter TNF‐induced apoptosis. Moreover, cells displaying more rapid apoptosis possess higher levels of type I 55 kDa TNFR1 receptor isoform, but less TNFR2. These findings reveal differential kinase activation in TNF‐induced apoptotic death.
Interaction of human cyclophilin hCyp‐18 with short peptides suggests the existence of two functionally independent subsites
Authors: Demange Luc , Moutiez Mireille , Vaudry Karine , Dugave Christophe
Published: October 2002

The binding of peptides, derived from the model substrate Suc‐Ala‐Ala‐Pro‐Phe‐pNA, to the human cyclophilin hCyp‐18 was investigated. HCyp‐18 is able to bind 2–4‐mer peptides as well as shorterpara‐nitroaniline (pNA) derivatives and pNA surrogates. Although Suc‐Ala‐Phe‐pNA binds hCyp‐18, only proline‐containing peptides are able to block efficiently the peptidyl‐prolylcis/transisomerase activity. Competition experiments strongly suggest the existence of two independent subsites: a S1′ ‘proline’ subsite and a S2′–S3′ ‘pNA’ subsite. The interaction at S2’–S3’ requires either a Phe‐pNA C‐terminus or a Phe‐pNA surrogate bearing an H‐bond acceptor able to bind Trp121 and Arg148 simultaneously.
Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A
Authors: Hatanaka Takahiro , Huang Wei , Martindale Robert G. , Ganapathy Vadivel
Published: October 2002

Treatment of HepG2 cells with forskolin led to 60–100% stimulation of system A activity, measured as the Na+‐dependent uptake of α‐(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non‐specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady‐state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA.
Activation of ADAM 12 protease by copper
Authors: Loechel Frosty , Wewer Ulla M.
Published: October 2002

Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12‐S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12‐S that was insensitive to copper. This suggests a multi‐step activation mechanism for ADAM 12 involving both furin cleavage and copper binding.
Refolding of β‐lactoglobulin studied by stopped‐flow circular dichroism at subzero temperatures
Authors: Qin Zhi-jie , Hu Dong-mei , Shimada Lui , Nakagawa Tatsuo , Arai Munehito , Zhou Jun-Mei , Kihara Hiroshi
Published: October 2002

Refolding of bovine β‐lactoglobulin was studied by stopped‐flow circular dichroism at subzero temperatures. In ethylene glycol 45%–buffer 55% at −15°C, the isomerization rate from the kinetic intermediate rich in α‐helix to the native state is approximately 300‐fold slower than that at 4°C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped‐flow apparatus (10 ms). At −28°C, we observed at least three phases; the fastest process, accompanied by an increase of α‐helix content, is completed within the dead time of the stopped‐flow apparatus (10 ms), the second phase, accompanied by an increase of α‐helix content with the rate of 2 s−1, and the third phase, accompanied by a decrease of α‐helix content. This last phase, corresponding to the isomerization process at −15°C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid α‐helix formation and their concurrent collapse are common even in proteins rich in β‐structure in their native forms.
Export of Thermus thermophilus alkaline phosphatase via the twin‐arginine translocation pathway in Escherichia coli
Authors: Angelini Sandra , Moreno Renata , Gouffi Kamila , Santini Claire-Lise , Yamagishi Akahiko , Berenguer José , Wu Long-Fei
Published: October 2002

The bacterial twin‐arginine translocation (Tat) pathway is distinct from the Sec system by its remarkable capacity to export folded enzymes. To address the question whether the two systems are capable of translocating homologous enzymes catalyzing the same reaction, we cloned the tap gene encoding Thermus thermophilus alkaline phosphatase (Tap) and expressed it in Escherichia coli. Unlike the alkaline phosphatase of E. coli, which is translocated through the Sec system and then activated in the periplasm, Tap was exported exclusively via the Tat pathway and active Tap precursor was observed in the cytoplasm. These results demonstrate that two sequence and functional related enzymes are exported by distinct protein transport systems, which may play an integral role in the bacterial adaptation to their environment during the evolution.
Toxicity of the bacterial luciferase substrate, n‐decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans
Authors: Hollis R.P. , Lagido C. , Pettitt J. , Porter A.J.R. , Killham K. , Paton G.I. , Glover L.A.
Published: October 2002

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n‐decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.
Elements from the cAMP signaling pathway are involved in the control of expression of the yeast gluconeogenic gene FBP1
Authors: Zaragoza Oscar , Gancedo Juana M
Published: October 2002

cAMP represses the transcription of some Saccharomyces cerevisiae genes sensitive to catabolite repression. The effect of cAMP on the expression of FBP1, encoding fructose‐1,6‐bisphosphatase (FbPase), has been further investigated. In yeast cells shifted to a derepressing medium, synthesis of FbPase was delayed if the strong decrease in intracellular cAMP, which occurs during the shift, was prevented. A similar delay occurred in a RAS2val19
strain, while in a tpk1w
strain, with weak protein kinase A activity, induction of FbPase occurred earlier than in a TPK1 strain. In the tpk1w
strain, proteins which bind the UAS1 element of FBP1 were present during growth on glucose but they were only weakly operative. Expression of CAT8 and SIP4, encoding proteins which bind the UAS2 element, was blocked by a high concentration of cAMP, but catabolite repression of these genes was not much relieved in a tpk1w
strain. We conclude that in S. cerevisiae, as reported for Schizosaccharomyces pombe, control of FBP1 requires both cAMP‐dependent and independent pathways; however, the mechanisms operating in the two yeasts are different.
Cysteine‐25 of adenylate kinase reacts with dithiothreitol to form an adduct upon aging of the enzyme
Authors: Li Xia , Han Yang , Pan Xian Ming
Published: October 2002

Adenylate kinase (AK) ages in solution in the presence of DL‐dithiothreitol (DTT) with a gradual activity decrease. Upon dilution with 4 M guanidine hydrochloride denatured native and aged AK, both recover to the same activity as the fresh enzyme. Mass spectroscopy and 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole chloride modification kinetics studies identify that the residue cysteine‐25 of the enzyme reacts with DTT to form an adduct. The formation of the unusual bridging DTT adduct of AK appears to be the result of a stable DTT–protein complex. The K
M for AMP, ADP and MgATP of the DTT‐modified enzyme does not differ significantly from that of the intact enzyme, whereas the secondary and tertiary structures of the enzyme change obviously. These results indicate that cysteine‐25 may not be involved directly in substrate binding, but may play an important role in maintaining secondary and tertiary structures of native AK, as well as the conformation interconversion in the catalytic cycle.
Transcriptional repression of the human p53 gene by cobalt chloride mimicking hypoxia
Authors: Lee Seok Geun , Lee Hyeon , Rho Hyune Mo
Published: October 2002

The switch to an angiogenic phenotype is known to be a fundamental determinant of neoplastic growth and tumor progression. We herein report that the transcription of the human p53 gene was repressed by treatment with a hypoxia‐mimicking concentration of cobalt chloride and alone by hypoxia‐inducible factor 1α. Analyses of serial deletions, site‐directed mutageneses and heterologous promoter systems showed that the site responsible for the repression by both factors was the E‐box element in the promoter of the p53 gene. These results alongside previous data suggest that the loss of p53 including the transcriptional repression may play an important role in the angiogenic switch during tumorigenesis.
An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA
Authors: Maeshima Hideki , Okuno Emi , Aimi Tadanori , Morinaga Tsutomu , Itoh Takuzi
Published: October 2002

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf‐SRP54) and the N‐terminal G‐domain and C‐terminal M‐domain (pf‐SRP54M) of pf‐SRP54 with an amino‐terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non‐conserved 213‐nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107‐nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine‐tagged pf‐SRP54M bound specifically to the conserved 107‐nucleotide RNA in the absence of pf‐SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.
Interleukin‐1β stimulates cyclic GMP efflux in brain astrocytes
Authors: Pedraza Carlos E , Baltrons Marı́a Antonia , Garcı́a Agustina
Published: October 2002

In rat brain astroglia‐enriched cultures long‐term treatment with interleukin‐1β induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin‐1β effect is mediated by type I receptor and potentiated by interferon‐γ. In cells treated with bacterial endotoxin a larger NO‐dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co‐treated with interleukin‐1β. The non‐selective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin‐1β stimulates a cGMP transporter.
Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion‐permeable channels in planar lipid bilayer membranes
Authors: Muto Yoshinori , Matsuoka Tatsuomi , Kida Akemi , Okano Yukio , Kirino Yutaka
Published: October 2002

Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation‐selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K+/Cl− permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismin's action against predatory protozoa.
Engineering of the H2O2‐binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2
Authors: Miyazaki Chie , Takahashi Haruo
Published: October 2002

The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn2+ to Mn3+, is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2‐binding pocket. Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non‐oxidizable Leu showed a great improvement. Furthermore, after treatment at 1 mM H2O2 where the wild‐type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non‐bulky and non‐oxidizable Ser.
Rapid increase of NO release in plant cell cultures induced by cytokinin
Authors: Tun Ni N , Holk André , Scherer Günther F.E
Published: October 2002

4,5‐Diaminofluorescein, a fluorescence indicator for NO, was applied to detect the release of NO from plant cells. NO production was increased within 3 min when plant cell cultures (Arabidopsis, parsley, and tobacco) were treated by cytokinin and was dose‐dependent and signal‐specific in that other plant hormones and inactive cytokinin analog were not effective in stimulating of NO release. The response was quenched by addition of 2‐(aminoethyl)‐2‐thiopseudourea, an inhibitor of the animal NO synthase, and by addition of an NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐1‐oxy‐3‐oxide. These results imply that NO may act in cytokinin signal transduction.
Clearance of group X secretory phospholipase A2 via mouse phospholipase A2 receptor
Authors: Yokota Yasunori , Notoya Mitsuru , Higashino Ken-ichi , Ishimoto Yoshikazu , Nakano Kazumi , Arita Hitoshi , Hanasaki Kohji
Published: October 2002

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A2 (sPLA2‐X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA2‐X can also act as a ligand for mouse phospholipase A2 receptor (PLA2R). Here, we found that sPLA2‐X was internalized and degraded via binding to PLA2R associated with the diminished prostaglandin E2 (PGE2) formation in PLA2R‐expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA2‐X was co‐localized with PLA2R in the punctate structures in PLA2R‐expressing CHO cells. Moreover, in mouse osteoblastic MC3T3‐E1 cells that endogenously express the PLA2R, the internalized sPLA2‐X was localized in lysosomes. These findings demonstrate that PLA2R acts as a clearance receptor for sPLA2‐X to suppress its strong enzymatic activity.